Colchicine binding to bovine anterior pituitary slices and inhibition of growth-hormone release.

The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1 +/- 0.69 mug. The binding capacity of these sites is 8.58 +/- 0.60 pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. Subcellular fractionation of colchicine-labelled slices shows that 90% of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 10(4) and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. The rate of colchicine uptake was studied at 0.84 mm- and 2mum-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10 min. Equilibration with the saturable binding sites was complete in 120 min at 2mum-colchicine. A concentration of colchicine (13.4 mum) which would give 81% maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37 degrees C in glutaraldehyde by 83 +/- 4%. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mm), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%.     

Reactivity patterns of monoclonal antibodies positive on myelomonocytic leukemia cells as defined by esterase isoenzyme analysis

Summary The reactivity with monoclonal antibodies (MoAbs) specific for myelomonocytic cells and the expression of a particular esterase isoenzyme were analyzed in 159 cases of acute myeloid leukemias. The incidence of positivity of 16 MoAbs (MCS-2, MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mo1, Mo2, 63D3) was studied using the indirect immunofluorescence technique. A carboxylic esterase isoenzyme which can be inhibited completely and selectively by sodium fluoride (NaF) was demonstrated by isoelectric focusing on horizontal polyacrylamide gels. This NaF-sensitive isoenzyme indicated the monocytic origin of the blast cells as it is specific for this cell lineage. Prior to the immunological-isoenzymatic analysis all cases were categorized into two subtypes according to morphological criteria of the FAB classification system: 147 cases of AML (FAB M1-3) and 12 cases of AMMoL/AMoL (FAB M4/5). However, 15 out of 147 cases of AML expressed the NaF-sensitive isoenzyme and were therefore assigned to the group AMMoL/AMoL. Likewise, 1 case, diagnosed morphologically as AMMoL, was negative for this marker isoenzyme and was assigned to the other leukemia subtype. The incidence of reactivity varied widely for the MoAbs tested regarding the overall results on all cases and the positivity on cases of either AML or AMMoL/AMoL. The MoAbs were grouped into four classes depending on the pattern of reactivity with myeloblastic or monoblastic or both subtypes of acute myeloid leukemia. The MoAbs MCS-2, MY7, Leu-M1, and MY9 detected the vast majority of cases with either myelocytic or monocytic involvement (group-I: pan-myelomonocytic reactivity). The MoAbs MCS-1, OKM1, VIM-D5, and Mo1 showed a predominance in their staining pattern for monocytic variants, but were also positive on a substantial percentage of nonmonocytic cases (group-II: predominantly reactive with monocytic, but also myelocytic cases). The MoAbs Leu-M3, MY4, VIM-D2, Mo2, and MY8 reacted with the large majority of AMMoL/AMoL cases and with a small number of AML cases (group-III: monocyte-specific reactivity). The MoAbs of group-I are useful in differentiating acute lymphoid from acute myeloid leukemias. The MoAbs of group-III, and to a lower extent those of group-II, will be of considerable value in the subtyping of acute myeloid leukemias. The results show that (1) accuracy of leukemia classification might not always be achieved by morphology alone, but that immunological and biochemical aspects should be included as well, and (2) several MoAbs are very useful tools for classification and subtyping of acute myeloid leukemias.     

Effect of minerals on glucose isomerase production by Streptomyces kanamyceticus

Summary A study has been made of the mineral requirements of Streptomyces kanamyceticus KCC S-0433 for production of glucose isomerase. The optimal concentrations of MgSO₄ and K₂HPO₄ for enzyme production are 0.07% and 0.05%, respectively. The elements Fe, Mn and Zn are required at levels of 10, 3 and 3 mg/l, respectively. Cu, Co and Ca have inhibitory effects on the production of the enzyme.     

Reduction of ferryl- and metmyoglobin to ferrous myoglobin by menadione-glutathione conjugate. Spectrophotometric studies under aerobic and anaerobic conditions

Both metmyoglobin (MbIII) and ferrylmyoglobin (MbIV) are reduced by the menadiol-glutathione conjugate (GS-Q2-) to oxymyoglobin (MbIIO2) or deoxymyoglobin (MbII), depending whether the assay is carried out under aerobic or anaerobic conditions, respectively. Under aerobic conditions, the reduction of MbIII to MbIIO2 by GS-Q2- is associated with O2 consumption. The latter process is accounted for by (a) the autoxidation of the conjugate yielding H2O2 and (b) the rapid binding of O2 to MbII to yield MbIIO2. The ratio [O2]consumed/[MbIIO2]formed is approximately 1.5 at the time when MbIIO2 formation is maximal (at about 0.8 min). This ratio, higher than the unit, indicates that there is more than one O2-consuming reaction in this experimental model. The ratio of initial rates of O2 consumption and MbIIO2 formation is close to the unit [(-dO2/dt)/(+ dMbIIO2/dt) = 1.1]. The formation of H2O2 originating during the autoxidation of the GS-Q2- is substantially lower in the presence of MbIII, probably due to the heterolytic cleavage of the O--O bond of the peroxide by the hemoprotein. Although the latter reaction should yield MbIV, this species is not observed in the absorption spectrum, probably due to its rapid reduction by GS-Q2-. MbIV is reduced to MbIIO2 by the GS-Q2-. Whether this reaction takes place in one-electron transfer steps, that is, the sequence: MbIV----MbIII----MbIIO2 is difficult to evaluate by absorption spectral analysis, due to the rapid rate of the [MbIV----MbIIO2] transition. Under anaerobic conditions, the reduction of either MbIII or MbIV by GS-Q2- yields MbII as a stable molecular product. Anaerobic conditions prevent any further interaction of MbII with intermediates of O2 reduction derived from GS-Q2- autoxidation.     

Gene family evolution across 12 Drosophila genomes

Comparison of whole genomes has revealed large and frequent changes in the size of gene families. These changes occur because of high rates of both gene gain (via duplication) and loss (via deletion or pseudogenization), as well as the evolution of entirely new genes. Here we use the genomes of 12 fully sequenced Drosophila species to study the gain and loss of genes at unprecedented resolution. We find large numbers of both gains and losses, with over 40% of all gene families differing in size among the Drosophila. Approximately 17 genes are estimated to be duplicated and fixed in a genome every million years, a rate on par with that previously found in both yeast and mammals. We find many instances of extreme expansions or contractions in the size of gene families, including the expansion of several sex- and spermatogenesis-related families in D. melanogaster that also evolve under positive selection at the nucleotide level. Newly evolved gene families in our dataset are associated with a class of testes-expressed genes known to have evolved de novo in a number of cases. Gene family comparisons also allow us to identify a number of annotated D. melanogaster genes that are unlikely to encode functional proteins, as well as to identify dozens of previously unannotated D. melanogaster genes with conserved homologs in the other Drosophila. Taken together, our results demonstrate that the apparent stasis in total gene number among species has masked rapid turnover in individual gene gain and loss. It is likely that this genomic revolving door has played a large role in shaping the morphological, physiological, and metabolic differences among species.     

A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells

We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.